Role Of TRPM8 Channels Activation In Regulation Of Store-Operated Calcium Entry (SOCE) In Pulmonary Artery Of Normoxic And Chronic Hypoxic Pulmonary Hypertensive Rats

Pulmonary hypertension?PH?is characterized by enhanced vasoconstriction and vascular remodeling,which are attributable to the alteration of Ca²⁺homeostasis in pulmonary arterial smooth muscle cells?PASMCs?.It is well established that store-operated Ca²⁺entry?SOCE?is augmented in PASMCs of idiopathic pulmonary hypertension?IPAH?patients and PH animal models and it plays crucial roles in PH development.Our previous studies showed that the melastatin-related transient receptor potential 8?TRPM8?was down-regulated in PASMCs of PH models;and activation of TRPM8 caused relaxation of pulmonary arteries?PAs?.However,the mechanism of TRPM8-induced vasorelaxation has not been determined.In this study,the interaction of TRPM8 and SOCE was examined in PAs and PASMCs of normoxic and chronic hypoxic pulmonary hypertensive?CHPH?rats.Firstly,we detected the alterations in the TRPM8 and canonical transient receptor potential 1?TRPC1?expression in PAs of CHPH rats,then to exam the effects of icilin-induced activation of TRPM8 on the SOCE-mediated vasoconstriction in rat pulmonary arterial rings,and to observe the effects of icilin on cyclopiazonic acid?CPA?-induced SOCE measured by Mn²⁺quenching of Fura-2 fluorescence and Ca²⁺transients in PASMCs of control and CHPH rats,finally,to observe the effects of icilin-induced activation of TRPM8 on the whole-cell store-operated cation current(I_SOC)in PASMCs of control rats and human.We further examined the interaction of TRPM8 and SOCE during the development of PH,in order to provide a new scientific basis for elucidating the pathogenesis of PH and its targeted therapy.1.The effect of icilin-induced activation of TRPM8 on the SOCE-mediated vasoconstriction in pulmonary arterial rings of normoxic and chronic hypoxic pulmonary hypertensive ratsObjective:To observe the alterations in the TRPM8 and TRPC1 expression in PAs of CHPH rats,and to observe the relaxant effects of icilin in CPA and Endothelin-1?ET-1?precontracted PAs of control and CHPH rats.Methods:On the basis of CHPH rats model induced by clean male Sprague-Dawley?SD?rats exposed to normobaric hypoxia for 3 weeks,using:?1?hemodynamics detection methods,right ventricle systolic pressure?RVSP?and mean pulmonary arterial pressure?mPPa?were measured using a Mikro-Tip pressure catheter approached through direct puncture of right ventricle?RV?,followed by advancing the catheter into the main PA using an open-chest approach,RV was separated from the left ventricle and septum?LV+S?,and right ventricular mass index?RVMI?;?2?quantitative real-time PCR and Western blot method,determination the alterations in the TRPM8 and TRPC1expression in PAs of CHPH rats;?3?immunofluorescent microscopy method,immunofluorescence analysis of TRPM8 and TRPC1 expression in PASMCs isolated from control and CHPH rats;?4?vascular ring tone detection methods,determination the relaxant effects of icilin and ET-1 in CPA and ET-1 precontracted PAs of control and CHPH rats.Results:In comparison with the control rats,?1?the RVSP,mP_Pa,and RVMI were markedly elevated in CHPH rats,indicated that significant PH had developed in the CHPH rat model;?2?TRPC1 mRNA and protein in isolated PAs of CHPH rats increased significantly,however,TRPM8 mRNA and protein expression was markedly decreased,suggested that the changes in the expression of TRPM8 and TRPC1 are opposite in the development of PH;?3?immunofluorescence analysis was performed to further characterize the expression of TRPM8 and TRPC1 proteins in PASMCs,the fluorescent intensity of TRPM8 was significantly suppressed,whereas the immunofluorescent signal of TRPC1 was increased robustly in PASMCs of CHPH rats,showed that TRPM8 and TRPC1 are co-expressed in PASMCs,TRPC1 expression is upregulated and TRPM8 expression is markedly decreased during CHPH;?4?CPA-induced endothelium-denuded PAs contraction was greater,icilin concentration-dependent relaxation of CPA pre-contracted PAs in control and CHPH rats,in the absence or presence of the TRPM8 channel antagonist AMTB,suggested that SOCE upregulation in PAs of CHPH rats,TRPM8 activation with icilin inhibits SOCE in PAs and the inhibition was enhanced during PH;?5?application of 10?M icilin caused significant relaxation of the CPA-induced contraction and the inhibition was enhanced during PH,addition of the SOCE blocker gadolinium(Gd³⁺)caused further relaxation,in contrast,application of Gd³⁺alone inhibited CPA-induced contraction to a level similar to that caused by icilin plus Gd³⁺,subsequent addition of icilin failed to cause further relaxation,suggested TRPM8 activation with icilin inhibited SOCE in PAs,in addition,the effect of icilin may dependent on the availability of SOCE;?6?ET-1-induced endothelium-denuded PAs contraction was greater,and the inhibition of icilin and Gd³⁺were enhanced during PH,the percentage inhibition induced by icilin and icilin plus Gd³⁺were significantly enhanced in CH PAs,moreover,application of Gd³⁺alone relaxed PAs of control rats and CHPH rats to a level similar to that of icilin+Gd³⁺,application of icilin in the presence of Gd³⁺failed to cause further relaxation,suggested that icilin inhibited SOCE activated by ET-1.Conclusion:Rats exposed to CH for 3 weeks developed PH and RV hypertrophy.TRPM8 and TRPC1 are co-expressed in PASMCs;TRPC1 expression is upregulated and TRPM8 expression is markedly decreased during CHPH.Since SOCE is important for the vascular remodeling and enhanced vasoconstriction in PH,down-regulation of TRPM8 in PASMCs may minimize its inhibitory influence to allow unimpeded SOCE activity for PH development.2.The effects of TRPM8 activation on CPA-induced cation entry and Ca²⁺transients in PASMCs of normoxic and chronic hypoxic pulmonary hypertensive ratsObjective:To observe the effects of TRPM8 activation on CPA-induced cation entry and Ca²⁺transients of PASMCs in control and CHPH rats,to further clarify the interaction of TRPM8 and SOCE,and heir functions in the pathogenic process of PH.Methods:On the basis of CHPH rats model induced by clean male SD rats exposed to normobaric hypoxia for 3 weeks,using:?1?Mn²⁺quenching of Fura-2 fluorescence detection method,icilin and CPA-mediated cation entry in PASMCs was assessed by Mn²⁺quenching of Fura-2 fluorescence,and observed the effect of icilin on CPA-induced SOCE in PASMCs of control and CHPH rats;?2?Fluo-3 fluorescence detection Ca²⁺transients methods,characterization of icilin and CPA-induced Ca²⁺transients in PASMCs of control and CHPH rats were assessed,and observed the effect of icilin on CPA-induced Ca²⁺transients in PASMCs of control and CHPH rats;?3?Fluo-3 fluorescence detection Ca²⁺transients methods,observed the effect of icilin on CPA-induced basic[Ca²⁺]_i and Ca²⁺influx in PASMCs of control and CHPH rats.Results:In comparison with the control rats,?1?icilin-induced the percent reduction of fluorescence signal and the maximal rate of Mn²⁺quenching were significantly reduced in CHPH PASMCs,in contrast,CPA-induced percent reduction of fluorescence signal and the maximal rate of Mn²⁺quenching were significantly increased in hypoxic PASMCs,suggested that the functional activity of TRPM8 and TRPC1 in PASMCs are altered in CHPH;?2?icilin attenuated the CPA-induced Mn²⁺quenching in PASMCs of control and CHPH rats,and the inhibitory effects of icilin was greater in PASMCs of CHPH rats compared to the control rats,suggested that TRPM8 activation with icilin inhibited SOCE in PASMCs;?3?TRPM8-induced inhibition of SOCE was further evaluated by measuring CPA-induced Ca²⁺transients,preincubation with icilin caused a reduction in the CPA-induced Ca²⁺influx,and the inhibitory effect of icilin on CPA-induced SOCE was more pronounced in CH PASMCs,moreover,the effect of icilin on CPA-induced Ca²⁺influx was completely blocked by AMTB in the control and CH cells,suggested that TRPM8 activation can effectively inhibit SOCE,and this effect is potentiated when SOCE is upregulated during CHPH;?4?preincubation with icilin caused a reduction in the baseline[Ca²⁺]_i,but had no effect on CPA-induced Ca²⁺release,however,the CPA-induced Ca²⁺influx was significantly reduced,and the inhibitory effects of icilin was greater in PASMCs of CHPH rats compared to the control rats,further suggested that TRPM8 activation with icilin inhibited SOCE in PASMCs;?5?Under normal baseline[Ca²⁺]_i,preincubation with icilin also caused a reduction in the CPA-induced Ca²⁺influx.Conclusion:The functional activity of TRPM8 and TRPC1 in PASMCs are altered in CHPH.TRPM8 activation can effectively inhibit SOCE;and this effect is potentiated when SOCE is upregulated during CHPH.3.The effects of icilin-induced activation of TRPM8 on the I_SOC in PASMCs of control rats and humanObjective:To observe the effects of icilin-induced activation of TRPM8 on the ISOC in PASMCs of control rats and human,to further clarify the interaction of TRPM8activation and SOCE.Methods:On the basis of control rats and human PASMCs,using:?1?the whole cell patch clamp technique,observed the effects of icilin-induced activation of TRPM8 and Gd³⁺on the CPA-induced I_SOC in PASMCs of control rats;?2?the whole cell patch clamp technique,observed the effects of icilin and Gd³⁺on the CPA-induced I_SOC in human PASMCs?HPASMCs?.Results:In PASMCs of control rats,?1?CPA induced increase of whole-cell I_SOCOC was suppressed by the SOCC channel blocker Gd³⁺,and suppressed inward and outward I_SOC in the presence of icilin?3?M and 10?M?,suggested that TRPM8 activation with icilin inhibited CPA-induced I_SOC in PASMCs of control rats;?2?application of 10?M icilin caused significant reduction of the CPA-induced I_SOC,and addition of Gd³⁺caused further reduction,suggested TRPM8 activation with icilin inhibited I_SOC in PASMCs,and the effect of icilin may dependent on the availability of SOCE;?3?CPA induced increase of whole-cell I_SOCOC was suppressed by the Gd³⁺,and suppressed inward and outward I_SOC in the presence of icilin in HPASMCs,and addition of Gd³⁺caused further reduction in the presence of icilin,further suggested that TRPM8 activation with icilin inhibited CPA-induced I_SOC in HPASMCs.Conclusion:In PASMCs of rats and HPASMCs,TRPM8 activation with icilin significantly inhibited the CPA-induced I_SOC.These results provide the direct evidence that TRPM8 activation can effectively inhibit SOCE.In summary,decreased expression and functional activity of TRPM8 and enhanced SOCE in PASMCs played an important role in the pathogenic process of CHPH.CH leads to PH and RV hypertrophy by up-regulating TRPC1 expression and SOCE activity.Since SOCE is important for the vascular remodeling and enhanced vasoconstriction in PH,down-regulation of TRPM8 in PASMCs may minimize its inhibitory influence to allow unimpeded SOCE activity for PH development.In addition,the activation of TRPM8 caused relaxation of PA through SOCE inhibition.In PASMCs of rats and HPASMCs,TRPM8 activation significantly inhibited the CPA-induced I_SOC.These results provide the direct evidence that TRPM8 activation can effectively inhibit SOCE.In conclusion,we have revealed a novel mechanism of TRPM8-dependent inhibition of SOCE in the pulmonary vasculatures,and down-regulation of TRPM8 may play a significant role in facilitating the development of PH.These findings may provide a new therapeutic target to prevent PH.